Spermatogenesis is a major testicular function that is regulated by an intertwined network of autocrine, endocrine and paracrine factors. The co-ordination of the gene regulatory networks, hormones and neurotransmitters is essential to establish a stable dynamic for cyclic proliferation and differentiation of spermatogonial stem cells (SSC) into mature spermatozoa. The homeostasis of testicular dynamics also requires a distinct immune environment (Rozwadowska et al., 2007). O’Bryan and Hedger, (2008) depict this as the “split immunological personality of the testes”, where the immune privilege of testes, courtesy of the blood-testes barrier, enables a tolerogenic environment for auto-immunogenic germ cells to sustain the normal progression of spermatogenesis while also providing efficient defense against pathogens (Kaur et al. 2013). This testicular immune-tolerance has been exploited to devise various approaches for germ cell transplantation, thus facilitating endangered species’ conservation, yield enhancement in aquaculture and transgenic organism engineering (Lacerda et al., 2013). Successful transplantation of donor primordial germ cell line has been demonstrated in blastula-phase recipient zebrafish and rainbow trout embryos where the endogenous development pathway of germ cells was blocked using dead end antisense morpholino oligos (Giraldez et al., 2005, Yoshizaki et al., 2016). However, the other facet of this split personality also involves the contribution of immune elements towards creating a suitable microenvironment for the normal progression of spermatogenesis (and not just tackling testicular pathologies). The seminiferous epithelium is reportedly a major site for secretion of inflammatory mediators that modulate testicular functions. The cyclical regulation of spermatogenesis by cytokines – viz. IL-1, IL-6 and TNF-α has been comprehensively studied in mammals and presents ample evidence of their implication in testicular physiology. All the three cytokines have been reported to modulate the dynamics of the blood-testes barrier for promoting the meiotic transition of leptotene/ preleptotene spermatocytes (Zhang et al., 2014). It is noteworthy that IL-1α production within testes is maintained constitutively and at maximal levels under normal conditions unlike IL-1β whose expression is relatively low in normal rat testis (O’Bryan and Hedger, 2008). Both IL-1 and IL-6 have been reported to act within an integrated network of local regulatory mechanisms at seminiferous epithelium (Zhou et al., 2019). Based on mammalian studies, it is hypothesized that the resorption of residual bodies post-spermiation (stage VIII-IX) may be responsible for triggering an inflammatory microenvironment at the seminiferous epithelium. IL-1α kickstarts spermatogonial proliferation for the next cycle of spermatogenesis while also inducing IL-6. The latter inhibits DNA synthesis and regulates the meiotic progression of preleptotene spermatocytes. Simultaneous stimulation of TNF-α initiates junction remodeling to mediate the transit of meiotic cells across the blood-testes barrier (Jin et al., 2010, O’Bryan and Hedger, 2008). Another cytokine, CSF-1 (colony stimulating factor-1), classically associated with hematopoiesis of immune cells and regulation of macrophage functions, has also been reported to promote self-renewal of SSCs in collaboration with GDNF (Glial cell line-derived neurotrophic factor) (Oatley et al., 2009, Sawaied et al., 2021). Additionally, various members of the chemokine family (CCL-2, CX3CR1) have also been implicated in regulation of spermatogonial renewal and differentiation in mammalian testis (Ramaswamy et al., 2017). Pattern recognition receptors like TLRs (toll-like receptors) and NLRs (NOD-like receptors) are also reported to be fundamental to regulation of Sertoli cell functions, apart from serving as defence sentinels within testis (Hedger, 2011, Tian et al., 2009). In a recent study by Yin et al., (2020), Nlrp14 emerged as prime candidate in governing the differentiation of primordial germ cell-like cell. According to the authors, Nlrp14 prevents the ubiquitination of HSPA2 which is a key requirement for DNA packaging in spermatids and consequent progression of spermatogenesis.

The above evidence suggests that the synchronicity of these inflammatory mediators at different timepoints of spermatogenic cycle is key to sustaining its continuity in mammals. However, in the most divergent vertebrate class- pisces, the role of inflammatory mediators in modulation of spermatogenesis under physiological conditions has not been widely explored. To the best of our knowledge, only a single study has been conducted to explore this aspect of fish reproduction where the authors studied the expression pattern of immune-relevant genes of Sparus aurata in testis and hypothesised a cyclical modulation of inflammatory mediators along the spermatogenic cycle of the teleost (Chaves-Pozo et al., 2008). In concurrence with the mammalian findings, the authors concluded that the maintenance of balance in the levels of immune mediators is crucial to driving a normal spermatogenic cycle. Nevertheless, beyond this report, further investigation of the non-canonical role of immune candidate genes in normal teleost testis presents an open avenue for research. The present study is a preliminary step to fulfilling this void wherein a comparative transcriptomics approach has been employed to segregate the reproduction-relevant and immune-relevant genes (nomenclature based on classical functions) from the DEG (differentially expressed genes) of spawning versus pre-spawning testis of Clarias magur, an important aquaculture candidate of the Indian sub-continent. Further, network-based analysis has been employed to highlight the enriched immune and reproductive functions being regulated across the two phases. The expression profiling of a few selected genes has been carried out across the four phases of testicular cycle and finally correlated with the testicular steroid profile. Our results reinstate the pivotal role of immune mediators in progression of normal spermatogenesis and highlight the primary gene pathways that mediate the transition from pre-spawning to spawning phase in C. magur.